Method for mammalian cell transfection using 25-hydroxycholesterol

ABSTRACT

The present invention relates to a process, a cell culture medium, and a kit for the rapid, high-efficiency transfection of mammalian cells with exogenous DNA. The process comprises incubating a cell culture in the presence of a transfection medium comprising a serum that is different from the serum in the normal growth medium used to grow the cells, and DNA to produce transfected cells. In a preferred embodiment the normal growth medium comprises fetal bovine serum and the transfection medium comprises a serum such as human, calf, horse, lamb, or pig serum. The transfection medium may further comprise an hydroxylated sterol such as 25-hydroxycholesterol.

This invention was made with government support under grant HL30568,awarded by The National Institutes of Health. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a method for introducing exogenous DNAinto mammalian cells by a rapid transfection method which is highlyefficient. A kit which provides the components for carrying out thetransfections of the present invention is also contemplated by thepresent invention.

BACKGROUND OF THE INVENTION

In the late 1970's, methods were developed which allowed theintroduction and expression of foreign or exogenous DNA into mammaliancells in culture. This technique, known as transfection ortransformation, is a powerful method for examining the function andexpression of various genes of mammals and many mammalian viruses.

By introducing exogenous DNA of interest into a cell, and monitoring theexpression of a gene contained in the DNA, the gene can be analyzed ingreater detail than in a cell where it is naturally expressed. Forexample, mutations and/or deletions of defined regions of a gene can beused to identify DNA elements within the gene that are important for theregulation of its expression. Such studies have lead to the discovery ofnovel transcriptional regulatory elements for genes as well as variousRNA processing and translational signals. In addition, the introductionof an exogenous gene into a cell line can be used to study the effect ofthat gene on the growth of the cell or the effect of drugs and otheragents on the expression of the transfected gene.

Transfection techniques have also been used to isolate genes bytransfecting cells with a pool of genomic DNA and selecting the desiredcells using genetic complementation techniques. Various mammalian genes,including the chicken thymidine kinase gene, the human hypoxanthineguanine phosphoribosyl-transferase (HPRT) gene, the human thymidylatesynthase gene, the human transferrin receptor gene, a human DNA repairgene, and new oncogenes, have been isolated using this technique.

The broad applicability and usefulness of transfecting DNA intomammalian cells has led to the development of a number of protocols forperforming transfections, many of which involve the use of eithercalcium phosphate or DEAE-dextran (or its analogs) as a carrier topromote the uptake of exogenous DNA by cultured mammalian cells. Othermethods have used "lipofection" techniques, which incorporate the use ofsynthetic cationic lipids to effect the transfection. Osmotic shock ofthe cells or treatment of the cells with lysosomal inhibitors has beenused in an attempt to enhance transfection efficiencies. Other attemptsto increase the efficiencies have used high-voltage electric pulses tocreate pores in the cell membranes to increase the efficiency of DNAuptake by the cells.

The transfection efficiencies obtained by these methods are relativelylow, ranging from 0.001% to 1%, depending on the cell line used as arecipient. (Transfection efficiency is often expressed as either: the %of cells which have acquired the characteristic conferred by theintroduced gene, as may be determined by staining the treated cells; ora measure of the aggregate amount of DNA taken-up by the cells asdetermined by assaying for a gene product encoded in the transfectedDNA.) These low transfection frequencies have limited the application ofthe technique to a few cell lines which exhibit high transfectionefficiencies.

The ability to transfect a wide variety of cell lines, such as thosecarrying mutations of interest, would facilitate the analyses of theregulatory regions controlling the expression of genes, allow theisolation of genes by genetic complementation, or the cloning of CDNAsequences on the basis of their expression.

Recently, there have been described higher-efficiency transfectiontechniques which use a modification of the calcium phosphate-mediatedtransfection method. The modified calcium phosphate-mediatedcell-transfection technique is performed in a BES(N,N-bis(2-hydroxyethyl)-2-amino-ethane-sulfonicacid)/phosphate-buffered saline, adjusted to a pH of about 6.9 to 7.0.An equal volume of the BES/phosphate-buffered saline is added to asolution comprising DNA and 0.25M CaCl₂, mixed, and incubated for 10 to20 minutes at room temperature. The DNA/CaPO₄ precipitate which forms isthen added dropwise to each 60 mm plate of cells incubated in 5 ml ofmedia containing fetal bovine serum (FBS). The cells are initiallyincubated in an atmosphere of 3%, by volume, CO₂ at 35° C. for about 15to 24 hours (Chen and Okayama, Molecular and Cellular Biology, 7,2745-2752, 1987). With this transfection method, cells are grown in 10%,by volume, FBS during the transfection procedure. Under theseconditions, many common cultured mammalian cell lines have reportedlybeen transfected with efficiencies as high as 10 to 50%. It should benoted that the results for determining transfection efficienciesreported from this method required long growth periods of the cellsprior to quantification, thus allowing "daughter" cells from theoriginally transfected cells to "reseed" the plate, resulting in anartificially high measure of transfection efficiency.

While such techniques represent an improvement over the previously knowntechniques, it is desirable that a transfection method is provided whichresults in higher transfection efficiencies, as determined by the numberof cells which take up DNA and the amount of DNA taken up by the cells.It is also desirable that the time taken to perform the transfectionprocedure is reduced so that results of the transfection can beevaluated in a shorter time period than is currently required.

In view of the foregoing, there is a need for a method of transfectingcells which results in high transfection efficiencies, with a widevariety of mammalian host cell lines, and which requires only a shortperiod of time for cells to take up exogenous DNA.

SUMMARY OF THE INVENTION

The present invention relates to a process for high-efficiencytransfection of mammalian cells. The process comprises incubating a cellculture in the presence of a transfection medium comprising a serum suchas human, calf, horse, lamb, or pig serum, and DNA to producetransfected cells, wherein the serum in the transfection medium isdifferent from the serum used to grow the cells.

More specifically, the process comprises growing mammalian cells in agrowth medium comprising a first serum such as FBS, then transferringthe cells to a transfection medium comprising a second serum, which isnot FBS, such as human, calf, horse, lamb, or pig serum, then adding acalcium phosphate-DNA precipitate solution to the medium over the cells,to form a calcium phosphate-DNA precipitate/medium mixture. Thetransfection medium may further comprise an hydroxylated sterol such as25-hydroxycholesterol.

The present invention also relates to a medium for transfectingmammalian cells which comprises a serum that is not FBS, such as human,calf, horse, lamb, or pig serum. The medium may further comprise anhydroxylated sterol such as 25-hydroxycholesterol.

Also contemplated by the present invention is a kit for the transfectionof mammalian cells comprising a transfection medium which comprises aserum that is not FBS, such as human, calf, horse, lamb, or pig serum.The kit may further comprise an hydroxylated sterol such as25-hydroxycholesterol.

DETAILED DESCRIPTION

In the present invention, cells are stably or transiently transfectedwith exogenous DNA under conditions which include the use of a calciumphosphate-DNA precipitate added to cells in a transfection medium. Thetransfection medium of the present invention comprises a serum that isdifferent from the serum used in the growth medium, which is used togrow the cells. During the transfection procedure, the cells areincubated at atemperature of about 35° C. to about 37° C., and in anatmosphere of about 2% to about 5%, by volume, CO₂. After a shortincubation period (about 3 to about 4 hours), during which the cellstake up the DNA, the cells are transferred to a growth medium and areincubatedat 37° C. in an atmosphere of about 5%, by volume, CO₂. In apreferred embodiment of the present invention, the transfection mediumcomprises about 10%, by volume, of a serum which is not FBS and about2.5 μg/ml of an hydroxylated sterol such as 25-hydroxycholesterol. Thesetransfection conditions result in a rapid and efficient uptake of theexogenous DNA by the host cell lines.

Transfection of Cells

Mammalian cells capable of growth in culture are suitable for use in thepresent invention. Preferably, the cells are adapted to growing inmonolayers. For use in the present invention, cells, growingexponentially, are harvested by trypsinization or by other techniquesthatare well known in the art. The harvested cells are then plated ontosterile60-mm plates and incubated until the cell cultures are about 20%to about 90% confluent. While 60-mm plates are preferred in the presentinvention (since they are easy to manipulate), other-sized plates couldalso be used. About 5 ml of a standard growth medium, such as Ham's F12medium comprising about 10% FBS, is added to the 60-mm plates containingthe cells, and the cells are incubated overnight (about 18 to about 20hours),at about 37° C. in an atmosphere of about 5%, by volume, CO₂, toallow them to attach to the surface of the plates.

The medium used to grow or transfect the cells is one that isappropriate for the cells being cultured. Such media are well known inthe art, and one of ordinary skill in the art would be familiar withmedia appropriate for growing the cells chosen to be cultured. Forexample, CV1 cells (American Type Culture Collection (ATCC) No. CCL70,Rockville, Md.) and mouse F9 cells (ATCC No. CRL1720) may be grown inDulbecco modified Eagle medium (DMEM), supplied by GIBCO of GrandIsland, N.Y.; CHO-K1 cells (ATCCNo. CCL61), HepG2 cells (ATCC No.HB8065), murine L cells (ATCC No. CCL1), and human Caco-2 cells (ATCCNo. HTB37) may all be grown in α-MEM orHam's F12 medium, supplied byGIBCO; and P3X63-AG8.653 cells (ATCC No. CRL1580) may be grown in RPMI1640 medium, also supplied by GIBCO.

Preferably, the normal growth medium used in accordance with the presentinvention comprises about 5% to about 20%, by volume, of a first serum,such as FBS supplied by Gemini of Calabasas, Calif. Most preferably, themedium comprises about 10%, by volume, FBS.

Antibiotics and fungicides may be added to the medium to prevent thegrowthof bacteria and fungi in the cell cultures. Antibiotics suitablefor use inthe practice of the present invention are those suchstreptomycin, penicillin, and fungizone (which are supplied byWhittacker Bioproducts ofWalkersville, Md., as a 100-fold concentratewhich is diluted 1:100 for use), although other antibiotics known in theart may also be used. These antibiotics may be added to all mediapreparations.

At the completion of the overnight incubation, the growth medium isremovedfrom the cells and the cells are washed about twice withphosphate bufferedsaline (PBS). A transfection medium comprising about5% to about 20%, by volume, of a second serum, which is different fromthe first serum, such as human, calf, horse, lamb, or pig serum, is thenadded to the cells. Preferably, if human serum is used in thetransfection medium, the blood from which the human serum is derived isdrawn from fasted individuals, toreduce the concentration of lipidspresent in the blood. The serum may be whole serum, lipoproteindeficient fraction of serum (LPDS), or other suitable serum fractions,serum components, or "synthetic serum" compositions. Most preferably,the transfection medium comprises about 10%, by volume, of the serum.

In a preferred embodiment of the present invention, the transfectionmediumalso comprises about 0.1 μg/ml to about 5 μg/ml of a hydroxylatedsterol to enhance the uptake of DNA by the cells. At a concentrationbelowabout 0.1 μg/ml, the beneficial effect of the hydroxylated sterolon increasing the efficiency of transfection is minimal; above aconcentration of about 5 μg/ml, little additional increase in theefficiency of transfection is observed. Most preferably, the mediumcomprises about 2.5 μg/ml of hydroxylated sterol. In the presentinvention 25-hydroxycholesterol is preferred.

A calcium phosphate-DNA precipitate solution is added to thetransfection medium. The calcium phosphate-DNA precipitate solution isprepared by mixing about 20 to about 30 μg of plasmid DNA with about 0.5ml of about 250 mM CaCl₂ (such as that supplied by Sigma Chemical Co. ofStLouis Mo., or J. T. Baker Chemical Co. Phillipsburg, N.J.). The CaCl₂solution is sterilized by filtering it through a filter such as a0.45-μm-pore-size nitrocellulose filter, supplied by Nalge of Rochester,N.Y., or by other suitable sterilization methods.

About 0.5 ml of a solution comprising about 50 mM of a buffer such asN,N-bis(2-hyroxyethyl)-2-aminoethane-sulfonic acid (BES) (supplied bySigma Chemical Co.), adjusted with HCl to about pH 6.9 to about 7.0,about280 mM NaCl, and about 1.5 mM Na₂ HPO₄ (sterilized as describedabove), is added to the DNA-CaCl₂ solution, and the resultant mixtureisincubated for about 10 to about 30 minutes at room temperature to formacalcium phosphate-DNA precipitate solution. The exact pH to be used istested for each BES solution prepared and for each cell line to betransfected.

The final concentration of the components in the calcium phosphate-DNAprecipitate solution is about 20 to about 30 μg/ml of plasmid DNA, about125 mM CaCl, about 25 mM BES, adjusted to a pH of about 6.9 to about7.0,about 140 mM NaCl, and about 0.75 mM Na₂ HPO₄. It is preferable that thetwo components of the mixture (i.e., the DNA-CaCl₂ and the BES-NaCl-Na₂HPO₄) are made up separatelyand then added together to promote formationof a calcium phosphate-DNA precipitate.

DNA for use in the present invention is preferably circular DNA, sincetransfection efficiencies are higher with circular DNA than with linearDNA. The DNA is preferably a plasmid which comprises the gene ofinterest,and may also comprise a marker gene, such as theβ-galactosidase gene (the E. coli lac Z gene). The inclusion of a markergene is convenient forassaying for transfection efficiency, since thepresence of the gene product, in this case β-galactosidase (β-gal), maybe easily assayed enzymatically or by staining. The relative activity ofthis enzymeproduced by the transfected cells is proportional to theefficiency of transfection.

Additionally, the plasmid may contain a gene which is a selectablemarker. For example, the "neo" gene (which confers resistance to thedrug G418) may be included in the transfection. The presence andexpression of such genes allows the selection of cells, which have takenup DNA, by growing them in a selection medium.

A gene to complement a mutation present in the mammalian host cells mayalso be used as a means of selecting particular transfectants. Suchcomplementation assays are well known to those skilled in the art.

The plasmid may also comprise a gene in the form of a cDNA copy of anydesired gene.

For use in the present invention, the genes to be transfected may beincorporated into a single plasmid or they may be incorporated intoseparate plasmids. Where the genes of interest are incorporated intoseparate plasmids, the plasmids are mixed and transfected together as asingle plasmid sample.

The construction of plasmids containing desired genes is well known tothose skilled in the art, and any such method of constructing plasmidsis suitable for use in the present invention.

Such plasmid DNA may then be grown in suitable host cells such asEscherichia coli (E. coli). The plasmid DNA may be isolated from thehost cells by any of the methods known in the art which result in thepurification of circular DNA. The purified plasmid is preferablyextractedextensively with phenol, chloroform, and ether, to ensure thatno E. coli proteins are present in the plasmid DNA preparation, sincethese proteins are often toxic to mammalian cells.

About 0.5 ml of the calcium phosphate-DNA precipitate solution is added,dropwise, to the transfection medium (comprising the second serum) oneachplate of cells to be transfected, and the mixture is swirled gently.The cells are then incubated for about 2 to about 4 hours, at about35°C., in an atmosphere of about 2% to about 4%, by volume, CO₂, andpreferably in an atmosphere of about 3%, by volume, CO₂, to allowthecells to take up the DNA from the calcium phosphate-DNA precipitate.

At the end of the incubation, the transfection medium comprising thesecondserum is removed and the cells are rinsed with PBS. Preferably,the rinsingis performed about twice. About 5 ml of the growth medium isadded to the cells, and the cells are then incubated for about 15-24hours, at about 37° C. in an atmosphere of about 5%, by volume, CO₂. Thecellsare then harvested and assayed for the presence of markergeneproduct.

The process of the present invention results in relative transfectionefficiencies that are as much as 20- to 2,000-fold higher than thoseobtained by other transfection procedures. The increased relativetransfection efficiency of the present invention appears, fromhistochemical studies, to result from an increase in the number of cellswhich take up DNA and from an increased amount of DNA taken up by thecells. Additionally, the time required for the uptake of the DNA isreduced to about 2 to about 4 hours, and protein produced by the markergenes transfected into the cells may be assayed within about 18 to about20 hours after the transfection procedure. In contrast, othertransfectionmethods require about 6 to about 24 hours for the cells totake up DNA, after which time a period of at least an additional 24 to72 hours is required before marker genes can be selected or assayed.Therefore, the process of the present invention results in a reductionof up to 48 hours in the total time required to perform a transfectionand to measure transfection efficiencies.

Transfection Kit

Also contemplated by the present invention is a kit for the transfectionofmammalian cells. Such a kit comprises the components required toconduct the transfection assay. Each of the components is supplied in aseparate container, as follows:

1. A concentrated transfection medium supplement, comprising about 100%,byvolume, serum that is not FBS, such as human, calf, horse, lamb, orpig serum. The supplement is diluted about ten-fold for use in medium.While asupplement requiring a 1:10 dilution is convenient for use in thepresent invention, lower concentrations, requiring lower dilution, mayalso be used.

2. A concentrated transfection medium supplement, comprising about 2.5mg/ml of an hydroxylated sterol such as 25-hydroxycholesterol inethanol. Preferably, the transfection medium is supplemented with about2.5 μg/ml 25-hydroxycholesterol. The concentrated supplement is diluted1,000-fold for use in medium. While a supplement requiring a 1:1,000dilution is convenient for use in the present invention, higher or lowerconcentrations, requiring higher or lower dilution, may also be used.However, since ethanol is toxic to the cells at high concentrations, itisdesirable that the final ethanol concentration in the transfectionmedium is relatively low.

3. A solution comprising sterile 250 mM CaCl₂. The CaCl₂ solutionmay besupplied as a sterile concentrate of about 2.5M, if preferred. If theCaCl₂ is supplied as a concentrate, it is diluted ten-fold for use. Aconcentrate may be preferred if large volumes of plasmid DNA are tobeused. Under these conditions, for one transfection experiment, thedilute plasmid DNA is added to a container, and about 0.05 ml of theconcentrated CaCl₂ is added. The volume of the mixture is then broughtto 0.5 ml by the addition of sterile, distilled water.

If concentrated solutions of plasmid DNA are used, however, the DNA maybe added directly to a diluted CaCl₂ sample, since the additionalvolumeof the DNA sample is insignificant.

4. A solution comprising sterile 50 mMN,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid (BES), about 280 mMNaCl, and about 1.5 mM Na₂ HPO₄. The pH of the solution is adjusted,prior to use, to a pH of about 6.9 to about 7.0.

5. Control plasmid DNA solutions, which are known to give hightransfectionefficiency in certain cell types, may be provided fortesting and comparingthe efficiency of transfection.

6. Assay reagents to assay the marker gene product incorporated into theplasmid DNA.

Kits prepared in accordance with the present invention may include allthe components listed above or only a few of the components which wouldnot normally be purchased by a laboratory conducting transfectionexperiments.However, the kit will include at least thetransfection-medium supplement comprising a serum that is not FBS, suchas human, calf, horse, lamb or pig serum.

EXAMPLE 1 Isolation of Plasmid DNA

The plasmid used in the following experiments was a β-gal-containingplasmid designated pON260, which comprises the cytomegalovirus promoterdescribed by Boshart et al. in Cell, 41, 521-531 (1985), the β-gal geneof E. coli, and the polyadenylation sequence from the SV40 late region,inserted into pBR322.

300 μl of an overnight culture of E. coli, which includes the pON260plasmid containing the β-gal gene, was added to 25 ml of Luria Broth andwas grown, at 37° C., to an optical density, at 550 nm, of 0.6.Ten ml ofthis culture was then added to one liter of Luria Broth supplementedwith ampicillin (100 μg/ml), and the culture was grown overnight, at 37°C., with shaking at 250 rpm.

The cells were collected by centrifugation at 7,000 rpm for 20 min. at4° C. The resultant pellet was resuspended in 80 ml of 10%(wt/vol)sucrose, 50 mM Tris-HCl, pH 8.0. Once the cells wereresuspended, 8 ml of 10 mg/ml lysozyme in 0.25 M Tris-HCl (pH 8.0), 16ml of water, and 16 ml of 0.5M EDTA were added to the cell suspension.The mixture was incubated on ice for 15 min. At the end of theincubation, 16 ml of 10% (wt/vol) SDSwas added, dropwise, as the cellsuspension was gently swirled. The solution was incubated on ice forabout 2 hours, with shaking. At the end of the incubation, the mixturewas centrifuged at 17,000 rpm for 15 min. at 4° C. The supernatant wascollected and filtered through cheesecloth, and 1 volume of 5M NaCl wasadded for each 4 volumes of supernant. The mixture was then incubated onice for 2 hrs., with shaking.Particulate matter was removed bycentrifugation at 13,000 rpm for 25 min. at 4° C., and the resultantsupernant was filtered through cheesecloth. The DNA contained in thefiltered supernatant was precipitated by adding 2 volumes of ethanol andincubating overnight at -70° C. The DNA precipitate was collected bycentrifugation at 8,000 rpm for 20 min. at 4° C.

The DNA pellet was resuspended in a final, total volume of 17 ml of 10mM Tris-HCl, pH 7.5, and 1 mM EDTA. 1.02 g of CsCl per ml of solutionwas added, as was 0.05 ml of 10 mg/ml of ethidium bromide, and thesolution was mixed until the CsCl was dissolved. The solution waschecked to ensurethat the refractive index after the addition of theCsCl and ethidium bromide was 1.396. The CsCl/ethidium bromide/DNAsolution was centrifuged at 35,000 rpm for 48 to 60 hrs. at 20° C. TheDNA band was collected and extracted four times with sec-butanol,saturated with CsCl, to remove the ethidium bromide from the DNA. Theextracted DNA was precipitated by adding one volume of water and fourvolumes of ethanol andincubating overnight at -20° C. The precipitatedDNA was collected by centrifugation at 8,000 rpm for 30 min. at 0° C.

The precipitate was resuspended in 10 mM Tris-HCl, pH 7.5, and 1 mM EDTAand extracted as follows: once with phenol, twice with phenol:chloroform(1:1), once with chloroform, and twice with ether. The extracted DNA wasprecipitated by adding a 0.5 volume of 7.5M ammonium acetate and 2.5volumes of ethanol and incubating overnight at -20° C. The precipitatewas collected by centrifugation at 8,000 rpm for 20 min. at 0° C., andwas resuspended in 400 μl of 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA.

The DNA was then reprecipitated by adding a 0.5 volume of 7.5M ammoniumacetate and 2.5 volumes of ethanol and incubating overnight at -20°C.The precipitate was collected by centrifugation at 8,000 rpm for 20min.at 0° C., and was resuspended in 500 μl of 10 mM Tris-HCl, pH 7.5,and 1 mM EDTA.

The plasmid DNA was then used in transfection experiments.

EXAMPLE 2 Control Transfection of CHO Cells with βgal Plasmid in thePresence of10% Fetal Bovine Serum in Ham's F12 Medium (Chen-OkayamaMethod)

Monolayers of CHO cells, growing exponentially in Ham's F12 mediumsupplemented with 10%, by volume, FBS were harvested by trypsinization.The cells were then plated in duplicate onto sterile 60-mm cultureplates and incubated in 5 ml of Ham's F12 medium supplemented with 10%,by volume, FBS, until the cell cultures were about 50% confluent.

After the incubation (usually 16-24 hrs.), the medium was removed, thecells were washed twice with PBS, and 5 ml of Ham's F12 mediumsupplemented with 10%, by volume, FBS was added to the cells.

25 μg of plasmid DNA, 8 μg of which was a β-gal-containing plasmid,prepared by the method described in Example 1, was mixed with 0.5ml of250 mM CaCl₂. 0.5 ml of 50 mM BES (pH 6.96), 280 mM NaCl, and 1.5 mM Na₂HPO₄ were added to the DNA-CaCl₂ solution, and the resultant mixture wasincubated, for not less than 10 but not more than 30 minutes, at roomtemperature, to form a calcium phosphate-DNA precipitate solution. Thetime difference in the incubation to form the calcium phosphate-DNAprecipitate solution is due to the time required to add the solution toeach of the individual cultures to be transfected as described below.

0.5 ml of the calcium phosphate-DNA precipitate solution was then added,dropwise, to each plate of cells, and the calcium phosphate-DNAprecipate/medium mixture was swirled gently. The plates were incubatedfor3 to 4 hours at 35° C. in an atmosphere of 3%, by volume, CO₂. At theend of the incubation, the Ham's F12 medium supplemented with 10% FBSwas removed and the cells were rinsed at least twice with PBS. Five mlofHam's F12 medium supplemented with 10%, by volume, FBS was added to thecells and the cells were incubated at 37° C. in an atmosphere of 5%, byvolume, CO₂. The following day (about 18 to 24 hours post-transfection),the cells were harvested, lysed by multiple freeze-thaw cycles, andcentrifuged at 10,000 rpm for 10 min. The supernatant was assayed forβ-gal activity.

The β-gal activity was assayed by adding 100 μl of supernatant of 900 μlof a solution comprising 100 mM sodium phosphate, pH 7.5, 10 mM KCl, 1mm MgSO₄, and 50 mM β-mercaptoethanol. After the mixturehad beenincubated at 37° C. for 5 min., 200 μl of 4 mg/ml O-nitrophenylβ-D-galactapyranoside, dissolved in 100 mm sodium phosphate (pH 7.5),was added and the solution was mixed. The solution wasincubated at 37°C. until a bright yellow color developed. At that time, the reaction wasterminated by the addition of 500 μl of 1M Ca₂ O₂. The adsorbance of thesamples was then read at 420 nm. The results are shown in Table I.

EXAMPLE 3 Transfection of CHO Cells with β-gal Plasmid in the Presenceof 5.8% Human Lipoprotein Deficient Serum (LPDS)

Monolayers of CHO cells, growing exponentially in Ham's F12 mediumsupplemented with 10%, by volume, FBS were harvested by trypsinization.The cells were then plated, to about 50% confluence, in duplicatesterile 60 mm culture plates and incubated overnight in 5 ml of Ham'sF12 medium supplemented with 10%, by volume, FBS.

After the incubation, the medium was removed, the cells were washedtwice with PBS, and 5 ml of Ham's F12 supplemented with 5.8% LPDS wasadded to the cells.

The remainder of the transfection procedure was as described in Example2. The results are shown in Table I.

EXAMPLE 4 Transfection of CHO Cells with β-gal Plasmid in the Presenceof 5.8% Human LPDS and 1 μg/ml of 25-Hydroxycholesterol/10 μg/mlCholesterolMixture (25-HC/C)

The procedure was the same as described in Example 3, except thetransfection was performed in the presence of Ham's F12 supplementedwith 5.8%, by volume, human LPDS, 1 μg/ml 25-hydroxycholesterol, and 10μg/ml cholesterol. The results are shown in Table I.

                  TABLE I                                                         ______________________________________                                                                          Rel.                                                                 β-gal                                                                             Transfec.                                   Example Medium           Activity.sup.1                                                                         Efficiency.sup.2                            ______________________________________                                        .sup. 2.sup.3                                                                         Ham's F12 + FBS    42      1                                          3       Ham's F12 + LPDS 1,375    33                                          4       Ham's F12 + LPDS +                                                                             3,462    82                                                  25-HC/C.sup.4                                                         ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS)                                                                .sup.3 control                                                                .sup.4 25hydroxycholesterol/cholesterol mixture                          

The results indicate that the addition of 5.8%, by volume, human LPDS inplace of the 10%, by volume, FBS normally used in transfectionprocedures,results in a 33-fold increase in relative transfectionefficiency. This efficiency is furthered to an 82-fold increase by alsosupplementing the medium with 1 μg/ml of 25-hydroxycholesterol and 10μg/ml cholesterol.

EXAMPLE 5 Transfection of CHO Cells with β-gal Plasmid Using 10% FBS inHam's F12 Medium (Control)

The procedure described in Example 2 was repeated. The results are showninTable II.

EXAMPLE 6 Transfection of CHO Cells with β-gal Plasmid Using 5.8% HumanLPDS in Ham's F12 Medium and 1 μg 25-hydroxycholesterol/ 10 μg/mlCholesterol (25-HC/C).

The procedure described in Example 4 was repeated. The results are showninTable II.

EXAMPLE 7 Transfection of CHO Cells with β-gal Plasmid Using 10% HumanSerum (HS) in Ham's F12 Medium

The procedure described in Example 3 was repeated except human serum wasused in place of LPDS. The results are shown in Table II.

EXAMPLE 8 Transfection of CHO Cells With β-gal Plasmid Using 10% HS inHam's F12Medium and 1 μg 25-hydroxycholesterol/ 10 μg/ml Cholesterol(25-HC/C)

The procedure described in Example 4 was repeated except that thetransfection was performed in the presence of Ham's F12 supplementedwith 10%, by volume, human serum, 1 μg/ml 25-hydroxycholesterol, and 10μg/ml cholesterol. The results are shown in Table II.

                  TABLE II                                                        ______________________________________                                                                          Rel.                                                                 β-gal                                                                             Transfec.                                   Example Medium           Activity.sup.1                                                                         Efficiency.sup.2                            ______________________________________                                        .sup. 5.sup.3                                                                         Ham's F12 + FBS   7        1                                          6       Ham's F12 + LPDS +                                                                             512      72                                                  25-HC/C.sup.4                                                         7       Ham's F12 + HS   386      54                                          8       Ham's F12 + HS + 1,034    146                                                 25-HC/C                                                               ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                                .sup.4 25hydroxycholesterol/cholesterol mixture                          

The results show that the use of human serum results in an increase inthe transfection efficiency over media supplemented with 10% FBS. Thishigh efficiency of transfection is further increased by the addition of1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol.

EXAMPLE 9 Effect of Incubation Time on Transfection Efficiency (Control)

The procedure described in Example 2 was repeated. The results are showninTable III.

EXAMPLE 10 Effect of Incubation Time on Transfection Efficiency

The procedure described in Example 7 was repeated, except the cells wereincubated in the presence of the calcium phosphate-DNAprecipitate/medium mixture for 1 hour instead of 3 hours. The resultsare shown in Table III.

EXAMPLE 11 Effect of Incubation Time on Transfection Efficiency

The procedure described in Example 7 was repeated except the cells wereincubated in the presence of the calcium phosphate-DNAprecipitate/medium mixture for 2 hours instead of 3 hours. The resultsare shown in Table III.

EXAMPLE 12 Effect of Incubation Time on Transfection Efficiency

The procedure described in Example 7 was repeated. The results are showninTable III.

EXAMPLE 13 Effect of Incubation Time on Transfection Efficiency

The procedure described in Example 7 was repeated except the cells wereincubated in the presence of the calcium phosphate-DNAprecipitate/medium mixture for 4 hours instead of 3 hours. The resultsare shown in Table III.

                  TABLE III                                                       ______________________________________                                                            Inc. time        Rel.                                                         w/calc.   β-gal                                                                           Transfec.                                Ex.   Medium        phos.-DNA Activity.sup.1                                                                       Efficiency.sup.2                         ______________________________________                                        .sup. Ham's F12 + FBS                                                                             3           12    1                                       10    Ham's F12 + HS                                                                              1           114   10                                      11    Ham's F12 + HS                                                                              2         1,326  111                                      12    Ham's F12 + HS                                                                              3         3,535  295                                      13    Ham's F12 + HS                                                                              4         4,090  241                                      ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                           

The results indicate that transfection is increased as the incubationtime,in the presence of the calcium phosphate-DNA precipitate/mediummixture, isincreased; although high levels of transfection are observedwith only 2 hours of incubation.

EXAMPLE 14 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency(Control)

The procedure described in Example 2 was repeated. The results are showninTable IV.

EXAMPLE 15 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 7 was repeated. The results are showninTable IV.

EXAMPLE 16 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 7 was repeated, except 0.1 μg/ml of25-hydroxycholesterol was added to the transfection medium in place ofthe1 μg/ml hydroxycholesterol/10 μg/ml cholesterol. The results areshown in Table IV.

EXAMPLE 17 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 16 was repeated, except 0.5 μg/ml of25-hydroxycholesterol was added to the transfection medium. The resultsare shown in Table IV.

EXAMPLE 18 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 16 was repeated, except 1.0 μg/ml of25-hydroxycholesterol was added to the transfection medium. The resultsare shown in Table IV.

EXAMPLE 19 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 16 was repeated, except 2.5 μg/ml of25-hydroxycholesterol was added to the transfection medium. The resultsare shown in Table IV.

EXAMPLE 20 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 16 was repeated, except 5.0 μg/ml of25-hydroxycholesterol was added to the transfection medium. The resultsare shown in Table IV.

EXAMPLE 21 Effect of Concentration of 25-Hydroxycholesterol onTransfection Efficiency

The procedure described in Example 16 was repeated, except 10.0 μg/mlof25-hydroxycholesterol was added to the transfection medium. Theresults areshown in Table IV.

                  TABLE IV                                                        ______________________________________                                                            25-                                                                           Hydroxy-         Rel.                                                         cholest.  β-gal                                                                           Transfec.                                Ex.   Medium        μg/ml  Activity.sup.1                                                                       Efficiency.sup.2                         ______________________________________                                        .sup. 14.sup.3                                                                      Ham's F12 + FBS                                                                             0            1.5  1                                       15    Ham's F12 + HS                                                                              0          76     51                                      16    Ham's F12 + HS                                                                                0.1     231    154                                      17    Ham's F12 + HS                                                                                0.5     518    346                                      18    Ham's F12 + HS                                                                              1         973    649                                      19    Ham's F12 + HS                                                                                2.5     1,385  923                                      20    Ham's F12 + HS                                                                              5         1,451  967                                      21    Ham's F12 + HS                                                                              10        1,465  977                                      ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                           

The results indicate that 2.5 μg/ml of 25-hydroxycholesterol added toHam's medium supplemented with 10% human serum results in optimumtransfection efficiencies.

EXAMPLE 22 Comparison of Different Transfection Methods (Control)

The procedure described in Example 2 was repeated. The results are showninTable V.

EXAMPLE 23 Comparison of Different Transfection Methods

The procedure described in Example 19 was repeated. The results areshown in Table V.

EXAMPLE 24 Comparison of Different Transfection Methods

The procedure described in Example 19 was repeated, except the humanserum used in the procedure was from a different donor. The results areshown inTable V.

EXAMPLE 25 Comparison of the Different Transfection Methods

Monolayers of CHO cells, growing exponentially in Ham's F12 mediumsupplemented with 10%, by volume, FBS were harvested by trypsinizing.The cells were then plated, to 50% confluence, in duplicate sterile60-mm culture plates and incubated overnight in 5 ml of Ham's F12 mediumsupplemented with 10%, by volume, FBS.

After the incubation, the medium was removed and the cells were washedtwice with 3 ml OPTIMEM (supplied by GIBCO), and 3 ml of OPTIMEM wasaddedto the cells.

15.0 μg of plasmid DNA, 4 μg of which was a β-gal-containing plasmid,prepared as described in Example 1, was diluted to 50 μl with water.Forty μg LIPOFECTIN (supplied by GIBCO) was diluted with an equal volumeof water. The DNA and LIPOFECTIN solutions were mixed, incubated for 15min. at room temperature, and added, dropwise, to each plate of cells.The LIPOFECTIN-DNA precipitate mixture was swirled gently,and the plateswere incubated for 6 hours at 37° C. in an atmosphereof 5%, by volume,CO₂. At the end of the incubation, the LIPOFECTIN-DNA precipitatemixture was removed. Five ml of Ham's F12 medium supplemented with 10%,by volume, FBS was added to the cells, and the cells were then incubatedfor 48 hours at 37° C. in an atmosphere of 5%, by volume, CO₂. The cellswere harvested, lysed by multiple freeze-thaw cycles, centrifuged, andassayed for β-gal activity. The β-gal assays were performed as describedin Example 2. The results are shown in Table V.

EXAMPLE 26 Comparison of Different Transfection Methods

The procedure described in Example 25 was repeated, except 35 μg ofLIPOFECTIN was added instead of 40 μg. The results are shown in Table V.

EXAMPLE 27 Comparison of Different Transfection Methods

The procedure described in Example 25 was repeated, except 30 μg ofLIPOFECTIN was added instead of 40 μg. The results are shown in Table V.

                  TABLE V                                                         ______________________________________                                                                           Rel.                                                                 β-gal                                                                             Transfec.                                  Example Medium            Activity.sup.1                                                                         Efficiency.sup.2                           ______________________________________                                        .sup. 22.sup.3                                                                        Ham's F12 + FBS     1.5     1                                         23      Ham's F12 + HS + 25-HC                                                                          3030     2020                                       24      Ham's F12 + HS + 25-HC                                                                          1236     824                                        25      Ham's F12 + FBS + 25       17                                                 40 μg/ml LIPOFECTIN                                                26      Ham's F12 + FBS + 47       31                                                 35 μg/ml LIPOFECTIN                                                27      Ham's F12 + FBS + 79       53                                                 30 μg/ml LIPOFECTIN                                                ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                           

Transfections in the presence of LIPOFECTIN were found to be lessefficientthan with Ham's F12 in the presence of 10% human serum and 2.5μg/ml 25-hydroxycholesterol.

EXAMPLE 28 Transfection with Human Hepatoma (Hep G2) Cells (Control)

The procedure described in Example 2 was repeated except human hepatomacells were used in place of CHO cells, the cells were grown in minimalessential media (MEM) (supplied by GIBCO), and 15 μg of plasmid DNA, 8μg of which was a β-gal-containing plasmid, was transfected into eachplate of Hep G2 cells. The results are shown in Table VI.

EXAMPLE 29 Transfection with Human Hepatoma (Hep G2) Cells

The procedure described in Example 3 was repeated, except human hepatomacells were used in place of CHO cells, the cells were grown in minimalessential media (MEM) (supplied by GIBCO), and 15 μg of plasmid DNA, 8μg of which was a β-gal-containing plasmid, was transfected into eachplate of Hep G2 cells. The results are shown in Table VI.

EXAMPLE 30 Transfection with Human Hepatoma (Hep G2) Cells

The procedure described in Example 4 was repeated, except human hepatomacells were used in place of CHO cells, the cells were grown in minimalessential media (MEM) (supplied by GIBCO), and 15 μg of plasmid DNA, 8μg of which was a β-gal-containing plasmid, was transfected into eachplate of Hep G2 cells. The results are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                                                           Rel.                                                                 β-gal                                                                             Transfec.                                  Example                                                                              Medium             Activity.sup.1                                                                         Efficiency.sup.2                           ______________________________________                                        .sup. 28.sup.3                                                                       MEM + FBS          0.53      1                                         29     MEM + LPDS         7.17     14                                         30     MEM + LPDS + 25-HC/C.sup.4                                                                       12.3     23                                         ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                                .sup.4 25hydroxycholesterol/cholesterol mixture                          

The results indicate that higher transfection efficiencies in Hep G2cells are obtained with the use of MEM supplemented with LPDS than withMEM supplemented with FBS. Increased transfection efficiencies areobtained when the medium is supplemented with 25-HC/C.

EXAMPLE 31 Transfection with Murine L Cells (Fibroblasts) (Control)

The procedure described in Example 2 was repeated, except murine L cellswere used in place of CHO cells. The results are shown in Table VII.

EXAMPLE 32 Transfection with Murine L Cells (Fibroblasts)

The procedure described in Example 3 was repeated, except murine L cellswere used in place of CHO cells. The results are shown in Table VII.

                  TABLE VII                                                       ______________________________________                                                                          Rel.                                                                 β-gal                                                                             Transfec.                                   Example   Medium         Activity.sup.1                                                                         Efficiency.sup.2                            ______________________________________                                        .sup. 31.sup.3                                                                          Ham's F12 + FBS                                                                              0.99      1                                          32        Ham's F12 + LPDS                                                                             22.1     22                                          ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS)                                                                .sup.3 control                                                           

The results indicate that higher transfection efficiencies in Murine Lcells are obtained with the use of Ham's F12 in the presence of 5.8%humanLPDS than in the presence of 10% FBS.

EXAMPLE 33 Comparison of FBS and LPDS on Transfection Efficiency Usingthe "Standard CaPO₄ Transfection Method"

CHO cells were transfected by the "standard CaPO₄ transfectionmethod"described in "Transfection DNA into Eukaryotic Cells" in: CurrentProtocolsin Molecular Biology, Vol. 1 (Ausubel et al., eds.), WileyInterscience, pp. 9.1.1-9.1.3, incorporated herein by this reference. Aβ-gal-containing plasmid, prepared by the method described in Example1,was used in the transfections, in which FBS was used in the growthmedium as well as in the transfection medium. The results are shown inTable VIII.

EXAMPLE 34 Comparison of FBS and LPDS on Transfection Efficiency Usingthe "Standard CaPO₄ Transfection Method"

The method of Example 33 was repeated, except LPDS was used in place ofFBSin the growth medium. The results are shown in Table VIII.

EXAMPLE 35 Comparison of FBS and LPDS on Transfection Efficiency Usingthe "Standard CaPO₄ Transfection Method"

The method of Example 33 was repeated, except LPDS was used in place ofFBSin the growth and transfection medium. The results are shown in TableVIII.

EXAMPLE 36 Comparison of FBS and LPDS on Transfection Efficiency Usingthe "Standard CaPO₄ Transfection Method"

The method of Example 33 was repeated, except LPDS was used in place ofFBSin the transfection medium. The results are shown in Table VIII.

                  TABLE VIII                                                      ______________________________________                                                  Transfec.     Growth   β-gal                                   Example   Serum         Serum    Activity.sup.1                               ______________________________________                                        .sup. 33.sup.2                                                                          FBS           FBS        36.1                                       34        FBS           LPDS       36.1                                       35        LPDS          LPDS     481                                          36        LPDS          FBS      582                                          ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 control                                                           

The results indicate that the increase in transfection efficiency is duetothe LPDS in the transfection medium. The serum used in the growthmedium has no effect on transfection efficiency. Addition of LPDS to thetransfection medium results in higher transfection efficiencies than FBSwhen the "standard CaPO₄ transfection method" is used.

EXAMPLE 37 Comparison of Different Hydroxylated Sterols (FBS Control).

The method of Example 2 was repeated. The results are shown in Table IX.

EXAMPLE 38 Comparison of Different Hydroxylated Sterols (HS Control)

The method of Example 7 was repeated. The results are shown in Table IX.

EXAMPLE 39 Comparison of Different Hydroxylated Sterols

The method of Example 7 was repeated, except 2.5 μg/ml of25-hydroxycholesterol was added to the transfection reaction. Theresults are shown in Table IX.

EXAMPLE 40 Comparison of Different Hydroxylated Sterols

The method of Example 7 was repeated, except 2.5 μg/ml of5-cholestan-3β-ol-7-one, supplied by Steraloids Inc. of Wilton, N.H.,wasadded to the transfection reaction. The results are shown in Table IX.

EXAMPLE 41 Comparison of Different Hydroxylated Sterols

The method of Example 7 was repeated, except 2.5 μg/ml of cholestan-3β,5α, 6β-triol, supplied by Steraloids Inc. ofWilton, N.H., was added tothe transfection reaction. The results are shownin Table IX.

EXAMPLE 42 Comparison of Different Hydroxylated Sterols

The method of Example 7 was repeated, except 2.5 μg/ml of 5β-cholanicacid-3α, 7α, 12-triol, supplied by Seraloids Inc. of Wilton, N.H., wasadded to the transfection reaction. The results are shown in Table IX.

EXAMPLE 43 Comparison of Different Hydroxylated Sterols

The method of Example 7 was repeated, except 2.5 μg/ml of 5β-cholanicacid-3,7,12-trione, supplied by Steraloids Inc. of Wilton, N.H., wasadded to the transfection reaction. The results are shown in Table IX.

                  TABLE IX                                                        ______________________________________                                                                            Rel.                                                                   β-gal                                                                           Transfec.                                 Ex.    Serum    Hydroxysterol                                                                              Activity.sup.1                                                                       Efficiency.sup.2                          ______________________________________                                        .sup. 37.sup.3                                                                       FBS      none         0.3     1                                        38     HS       none         23.4   78                                        39     HS       25-HC.sup.4  55.9   186                                       40     HS       C-7-one.sup.5                                                                              25.6   85                                        41     HS       C-triol.sup.6                                                                              15.8   53                                        42     HS       CA-triol.sup.7                                                                             29.5   98                                        43     HS       CA-trione.sup.8                                                                            16.9   56                                        ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                                .sup.4 25hydroxycholesterol                                                   .sup.5 5cholestan-3ol-7-one                                                   .sup.6 cholestan3β,6α,6triol                                       .sup.7 5cholanic acid3α,7α,12triol                                .sup.8 5cholanic acid3,7,12-trione                                       

The results indicate that significant enhancement of the transfectionefficiency is obtained with 25-hydroxycholesterol.

EXAMPLE 44 Effect of Sera from Different Animals on TransfectionEfficiency

The procedure described in Example 2 was repeated. The results are showninTable X.

EXAMPLE 45 Effect of Sera from Different Animals on TransfectionEfficiency

The procedure described in Example 19 was repeated. The results areshown in Table X.

EXAMPLE 46 Effect of Calf Serum on Transfection Efficiency

The procedure described in Example 19 was repeated, except 10% calfserum (supplied by GIBCO) was used in place of the human serum. Theresults are shown in Table X.

EXAMPLE 47 Effect of Newborn Calf Serum on Transfection Efficiency

The procedure described in Example 19 was repeated, except 10% newborncalfserum (supplied by GIBCO) was used in place of the human serum. Theresultsare shown in Table X.

EXAMPLE 48 Effect of Lamb Serum on Transfection Efficiency

The procedure described in Example 19 was repeated, except 10% lambserum (supplied by GIBCO) was used in place of the human serum. Theresults are shown in Table X.

EXAMPLE 49 Effect of Horse Serum on Transfection Efficiency

The procedure described in Example 19 was repeated, except 10% horseserum (supplied by GIBCO) was used in place of the human serum. Theresults are shown in Table X.

EXAMPLE 50 Effect of Chicken Serum on Transfection Efficiency

The procedure described in Example 19 was repeated, except 10% chickenserum (supplied by GIBCO) was used in place of the human serum. Theresults are shown in Table X.

EXAMPLE 51 Effect of Pig Serum on Transfection Efficiency

The procedure described in Example 19 was repeated, except 10% pig serum(supplied by GIBCO) was used in place of the human serum. The resultsare shown in Table X.

                  TABLE X                                                         ______________________________________                                                                        Rel.                                                                 β-gal                                                                             Transfec.                                     Ex.      Serum         Activity.sup.1                                                                         Efficiency.sup.2                              ______________________________________                                        .sup. 44.sup.3                                                                         Fetal bovine    0.3     1                                            45       Human.sup.4   103      343                                           46       Calf.sup.4    155      517                                           47       Newborn calf.sup.4                                                                          49       165                                           48       Lamb.sup.4    89       297                                           49       Horse.sup.4   115      382                                           50       Chicken.sup.4 10        34                                           51       Pig.sup.4     89       269                                           ______________________________________                                         .sup.1 nmoles of onitrophenol formed per min. per mg of protein               .sup.2 relative transfection efficiency is calculated by dividing the         units of gal activity in the test sample by the units of gal activity in      the control (the transfection performed with medium supplemented with 10%     by volume FBS).                                                               .sup.3 control                                                                .sup.4 2.5 μg/ml 25hydroxycholesterol was included in the transfection     experiment                                                               

The results indicate that significant enhancement over the controltransfection efficiency is obtained with human, calf, lamb, horse andpig sera. Higher transfection efficiencies where obtained with newborncalf and chicken sera than were obtained with FBS.

The above descriptions of exemplary embodiments of methods for improvedmammalian cell transfection are for illustrative purposes. Because ofvariations which will be apparent to those skilled in the art, thepresentinvention is not intended to be limited to the particularembodiments described above. The present invention may also be practicedin the absence of any element not specifically disclosed. The scope ofthe invention is defined by the following claims.

What is claimed is:
 1. A process for transfecting a mammalian cell withexogenous DNA comprising:growing a mammalian cell in a growth mediumcomprising fetal bovine serum; replacing the growth medium comprisingfetal bovine serum with a transfection medium comprising a second serumsupplemented with 25-hydroxycholestrol, where the second serum is notfetal bovine serum; adding a calcium phosphate-DNA precipitate solutionto the transfection medium over the cell to form a calcium phosphate-DNAprecipitate/medium mixture; washing the cell; and incubating the cell inthe presence of a growth medium.
 2. A process as recited in claim 1wherein the fetal bovine serum is present at a concentration of 5% to20%, by volume, of the growth medium.
 3. A process as recited in claim 1wherein the second serum is present at a concentration of 5% to 20%, byvolume of the transfection medium.
 4. A process as recited in claim 1wherein the 25-hydroxycholesterol is present at a concentration of 0.1μg/ml to 5 μg/ml.
 5. A process as recited in claim 1 wherein the25-hydrocholesterol sterol is present at a concentration of 1 μg/ml to 5μg/ml.
 6. A process as recited in claim 1 wherein the25-hydroxycholesterol is present at a concentration of 2.5 μg/ml.
 7. Aprocess as recited in claim 1 wherein the calcium phosphate-DNAprecipitate solution comprises:125 mM calcium chloride; 25 mMN,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid, pH 6.9 to 7.0; 140mM sodium chloride; and 0.75 mM sodium phosphate.
 8. A process asrecited in claim 1 wherein the cell is incubated in the presence of thecalcium phosphate-DNA precipitate/medium mixture for 1 to 6 hours.
 9. Aprocess as recited in claim 1 wherein the cell is incubated in thepresence of the calcium phosphate-DNA precipitate/medium mixture for 3to 4 hours.
 10. A process for transfecting a mammalian cell withexogenous DNA comprising:growing a mammalian cell culture in a mediumsupplemented with fetal bovine serum; removing the medium and supplementfrom the cell; adding a transfection medium supplemented with 10%, byvolume, of a serum and 2.5 μg/ml 25-hydroxycholesterol to a mammaliancell culture, wherein the serum is selected from the group consisting ofhuman, newborn calf, calf, horse, lamb, and pig sera; adding a calciumphosphate-DNA precipitate solution to the transfection medium over thecell to form a calcium phosphate-DNA precipitate/medium mixture;incubating the cell in the presence of the calcium phosphate-DNAprecipitate/medium mixture at 35° C. to 37° C. in an atmosphere of 3% to5%, by volume, carbon dioxide for less than 6 hours; washing the cell;and incubating the cell in the presence of medium supplemented withfetal bovine serum at 37° C. in an atmosphere of 5%, by volume, carbondioxide.